Self-applied prophylactic agents to prevent mucosal, particularly vaginal or rectal, transmission of HIV-1 have the advantage of empowering vulnerable receptive partners to take effective measures for their own protection. In our search for molecules that would be ideal templates for microbicide development, we utilized solid-phase peptide synthesis to recreate an evolutionary lost human antimicrobial peptide "retrocyclin" from its remains, a theta-defensin pseudogene. The ability of retrocyclin to potently prevent infection of CD4+ cells by both X4 and R5 HIV-1 was remarkable. Additional studies revealed that retrocyclin inhibits the initial steps in HIV-1 binding and entry as its mode of action. Next-generation analogs of retrocyclin were found to be active against numerous primary isolates from most groups and subtypes of HIV-1. Importantly, we have identified a lead compound, RC-101, that is highly active against HIV-1, non-cytotoxic, and suitable for preclinical development as a topical microbicide. Based on our studies, we have formed several hypotheses about retrocyclins: A) retrocyclins will likely function to inhibit HIV-1 infection in the vaginal mucosa, B) the glycosylated targets of retrocyclins and cyanovirin N, another potent anti-HIV-1 lectin (Project 1), are different and thus their activities may be complementary, and C) retrocyclins are potent antiretroviral agents suitable for further development as topical vaginal microbicides to prevent HIV transmission. To test these hypotheses, we propose to: 1) Construct and characterize next-generation analogs of anti-HIV retrocyclins, 2) Evaluate candidate retrocyclin formulations for anti-HIV-1 activity, stability and cytotoxicity, and 3) Evaluate candidate retrocyclin formulations for biologic efficacy in human vaginal fluid and serum. While it may be speculative to assert that the evolutionary loss of retrocyclin contributed to the susceptibility of humans to HIV-1 infection, retrocyclins are promising leads for designing naturally occuring microbicides that can prevent HIV infections.